CTLA4 fusion proteins for the treatment of diabetes

ABSTRACT

A method of treating, preventing, or delaying the progression of Type 1 diabetes mellitus autoimmunity by administering an effective amount of a cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) molecule is provided herewith. The CTLA4 molecule may be a fusion protein of a CTLA4 extracellular region and an immunoglobulin, such as abatacept.

PRIORITY

This application claims priority to U.S. patent application Ser. No.14/021,629 filed Sep. 9, 2013, which claims priority to U.S. patentapplication Ser. No. 13/534,560 filed Jun. 27, 2012, herein incorporatedby reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to the field of autoimmunedisease and specifically to the treatment, prevention, or delayedprogression of Type 1 diabetes mellitus.

BACKGROUND

The most common form of Type 1 diabetes mellitus (T1DM) is animmune-mediated disease where insulin-secreting β-cells are destroyed byan autoimmune response. There are a number of genetic and environmentalfactors associated with the onset of the disease, which involves theprogressive inflammatory infiltration of pancreatic islets containingimmunocytes targeted specifically to insulin-secreting β-cells. Thispathology develops over an indeterminate period of time (months toyears). While the discovery of insulin allowed for the treatment ofT1DM, there is currently no cure. The most common form of Type 1diabetes mellitus is immune-mediated, in which insulin-producing β cellsare destroyed. Yet, at the time of diagnosis, most patients still haveappreciable amounts of insulin production. Preservation of residualβ-cell function is highly desirable because it can reduce short-term andlong-term complications of the disease.

Several clinical trials have been undertaken in an attempt to arrestautoimmunity in Type 1 diabetes with immunomodulatory agents orantigen-based treatments. Most notably, trials of anti-CD3, anti-CD20,and a GAD-65 antigen vaccine have shown some efficacy in preservation ofβ-cell function as evidenced by stimulated C-peptide secretion. T cellsplay a central part in autoimmunity associated with Type 1 diabetes.

However, there is need for additional new therapies for Type 1 diabetesmellitus that are able to halt or slow autoimmune β-cell destructionleading to preservation of β-cell function and C-peptide secretion,particularly in patients recently diagnosed with Type 1 diabetes.

SUMMARY

In accordance with certain embodiments of the present invention relateto a method of treating diabetes mellitus in a subject comprisingadministering an effective amount of a fusion protein compositioncomprising a T-cell co-stimulation antagonist and a portion of animmunoglobulin molecule.

In some embodiments, the T-cell co-stimulation antagonist comprises theextracellular domain of CTLA4, an effective fragment of theextracellular domain or immunologically active variant of theextracellular domain. The T-cell co-stimulation antagonist may bind a B7antigen expressed on B cells or other antigen presenting cells (APCs).In some embodiments, the B7 antigen is expressed on B cells and on APCs.

In some embodiments, the fusion protein is Abatacept. In someembodiments, the composition further comprises an oil-based carrier suchas a water-in-oil emulsion (e.g., IFA or Montamide ISA). The compositionmay be administered by intravenous infusion, such as in about 50 to 200ml of physiological saline or at a dose ranging from about 5 mg/kg toabout 50 mg/kg or at a dose ranging from about 250 to 2000 mg, or at adose of 500 mg, 750 mg, or 1000 mg.

The methods as described herein may also comprise determining levels ofC-peptide in blood samples taken from the subject over time as anindicator of effectiveness of the treatment in inhibiting activation ofauto-aggressive T-cells. In some embodiments, the effectiveness of thecomposition in inhibiting activation of auto-aggressive T-cells isindicated by maintenance of C-peptide production or a delay in reductionof C-peptide production as compared to a standard or by improved HbA1cor reduction in the use of insulin by said subject as compared to astandard. Such measurements can be done ex vivo, such as by analyzing ablood sample. The reduction of C-peptide production in said subject maybe delayed for at least 3, 6, 9, 12, or 18 months, or 2, 3, 4, or moreyears.

In some embodiments, a preferred patient population is treated. Forexample, a patient selected from a population having a statisticallygreater response rate, such as a white patient, is treated.

Some embodiments provided herewith provide a method of preventing theonset of diabetes in a subject at risk for diabetes mellitus comprisingadministering an effective amount of a fusion protein compositioncomprising a T-cell co-stimulation antagonist and a portion of animmunoglobulin molecule. Some embodiments provided herewith provide amethod delaying the onset of diabetes by at least 3, 6, 9, 12, or 18months, or 2, 3, 4, or more years in a subject at risk for diabetesmellitus by administering an effective amount of a fusion proteincomposition comprising a T-cell co-stimulation antagonist and a portionof an immunoglobulin molecule.

These and other features of the embodiments as will be apparent are setforth and described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of various embodiments is provided herein belowwith reference, by way of example, to the following drawings. Theskilled person in the art will understand that the drawings, describedbelow, are for illustration purposes only. The drawings are not intendedto limit the scope of the applicant's teachings in any way.

FIG. 1 is the population mean of stimulated C-peptide 2-h AUC mean overtime for each treatment group. The estimates are from the ANCOVA modeladjusting for age, sex, baseline value of C-peptide, and treatmentassignment. Y-axis is on a log(y+1) scale. Error bars show 95%confidence intervals (CIs). AUC=area under the curve.

FIG. 2 is the predicted population mean of stimulated C-peptide 2-h AUCmean over time for each treatment group. Estimates are from the analysisof mixed-effects model adjusting for age, sex, baseline value ofC-peptide, and treatment assignment, and including a fixed effect fortime as a linear line on the log(y+1) scale. AUC=area under the curve.

FIG. 3 is the proportion of participants with 2-h peak C-peptideremaining at or above 0.2 nmol/L over time for each treatment group.

FIGS. 4A and 4B are the population mean of (A) HbA1c and (B) insulin useover time for each treatment group. Estimates are from the ANCOVA modeladjusting for age, sex, baseline value of HbA1c, and treatmentassignment. Insulin use is per kg of bodyweight, at 3-month intervals.Error bars show 95% CIs. HbA1c is glycated haemoglobin A1c.

FIG. 5 is the ratio (abatacept to placebo) of treatment effect on 2-yearstimulated C-peptide AUC mean within categories of prespecified baselinefactors. Estimates are from the ANCOVA modeling log of C-peptideadjusting for age, sex, baseline value of C-peptide, the indicatedcategorized factor, treatment assignment, and treatment interactionterms. The homogeneity test of treatment effect was significant for DR3allele status (p=0.025) and race (p=0.0003). AUC=area under the curve.HbA1c=glycated haemoglobin A1c.

It will be understood that the drawings are exemplary only and that allreference to the drawings is made for the purpose of illustration only,and is not intended to limit the scope of the embodiments describedherein below in any way.

DETAILED DESCRIPTION

It has been found that a CTLA4 molecule can be used for the treatment,prevention, or delayed progression of Type 1 diabetes mellitus (T1DM) ina subject.

Preservation of residual β-cell function (as measured by peak C-peptide≥0.2 nmol/L) is highly desirable because it can reduce short-term andlong-term complications of the disease. Several clinical trials havebeen undertaken in an attempt to arrest autoimmunity in Type 1 diabeteswith immunomodulatory agents or antigen-based treatments. Most notably,trials of anti-CD3, anti-CD20, and a GAD-65 antigen vaccine have shownsome efficacy in preservation of β-cell function as evidenced bystimulated C-peptide secretion. C-peptide is a protein that is producedin the body along with insulin. In a healthy pancreas, preproinsulin issecreted with an A-chain, C-peptide, a B-chain, and a signal sequence.The signal sequence is cut off, leaving proinsulin. Then the C-peptideis cut out, leaving the A-chain and B-chain to form insulin. SinceC-peptide and insulin are present in equimolar amounts, it is a highlyreliable marker for insulin production and the health of pancreatic βcells.

T cells play a central part in autoimmunity associated with Type 1diabetes. To become fully activated and autoaggressive, T cells arebelieved to need at least two crucial signals. (Marelli-Berg F M,Okkenhaug K, Mirenda V. A Trends Immunol 2007; 28: 267-73.) The firstsignal is an interaction between an antigen in the groove of the MHCmolecule on antigen-presenting cells and the T-cell receptor (TCR). Themost important second signal is the interaction between CD80 and CD86 onthe antigen presenting cells (APCs) and CD28 on the T cells. Thiscostimulatory second signal is needed for full activation of cells, andwithout it T cells do not become functional. Therefore, co-stimulationblockade has been proposed as a therapeutic modality for autoimmunityand transplantation. (Bluestone J A, St Clair E W, Turka L A. Immunity2006; 24: 233-38.)

Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4), which is also knownas CD152, is a protein involved in the regulation of the immune system.Naturally occurring CTLA4 is described in U.S. Pat. Nos. 5,434,131,5,844,095, and 5,851,795. Natural CTLA4 proteins are encoded by theCTLA4 gene. CTLA4 is a cell surface protein, having an N-terminalextracellular domain, a transmembrane domain, and a C-terminalcytoplasmic domain. The extracellular domain binds to and/or interfereswith target antigens, such as CD80 and CD86, serves as nature naturalbreak of T cell stimulation. In some embodiments, the extracellulardomain of the CTLA4 molecule begins with methionine at position +1 andends at aspartic acid at position +124; in other embodiments, theextracellular domain begins with alanine at position −1 and ends ataspartic acid at position +124.

A CTLA4 molecule is a molecule comprising a cytotoxicT-lymphocyte-associated antigen 4 (CTLA4) extracellular domain. In someembodiments, the extracellular domain of CTLA4 comprises a portion ofthe CTLA4 protein that recognizes and binds to at least one B7 (CD80/86)antigens such as a B7 antigen expressed on B cells and APCs. Theextracellular domain may also include fragments or derivatives of CTLA4that bind a B7 antigen. The CTLA4 extracellular domain can alsorecognize and bind CD80 (B7-1) and/or CD86 (B7-2). The extracellulardomain may also include fragments or derivatives of CTLA4 that bind abinds CD80 and/or CD86.

The CTLA4 molecule may be a fusion protein, where a fusion protein isdefined as one or more amino acid sequences joined together usingmethods well known in the art. The joined amino acid sequences therebyform one fusion protein. In some embodiments, the CTLA4 moleculecontains at least a portion of an immunoglobulin, such as the Fc portionof an immunoglobulin. In some embodiments, the CTLA4 molecule is anisolated and purified CTLA4 molecule.

In some embodiments, the CTLA4 molecule is a protein containing at leasta portion of an immunoglobulin, such as the Fc portion of animmunoglobulin. In some embodiments, the CTLA4 molecule is an isolatedand purified CTLA4 molecule.

In one preferred embodiment, the CTLA4 molecule is abatacept. Abataceptis a soluble fusion protein that consists of the extracellular domain ofhuman CTLA-4 linked to the modified Fc (hinge, CH2, and CH3 domains)portion of human immunoglobulin G1 (IgG1). Abatacept is produced byrecombinant DNA technology in a mammalian cell expression system. Theapparent molecular weight of abatacept is 92 kilodaltons.

Abatacept was developed for use in adult rheumatoid arthritis andjuvenile idiopathic arthritis and is indicated for reducing signs andsymptoms, inducing major clinical response, inhibiting the progressionof structural damage, and improving physical function in adult patientswith moderately to severely active rheumatoid arthritis.

Abatacept was developed by Bristol-Myers Squibb and is disclosed, forexample, in U.S. Pat. No. 5,851,795, U.S. Pat. No. 7,455,835, and U.S.Pat. Pub. 20011/311529. Abatacept, under the trade name ORENCIA, may beused as monotherapy or concomitantly with disease-modifyingantirheumatic drugs (DMARDs) other than tumor necrosis factor (TNF)antagonists. Abatacept is also indicated for reducing signs and symptomsin pediatric patients 6 years of age and older with moderately toseverely active polyarticular juvenile idiopathic arthritis. Abataceptmay be used as monotherapy or concomitantly with methotrexate (MTX).Since abatacept is a selective costimulation modulator and inhibits thecostimulation of T cells, it should not be administered concomitantlywith TNF antagonists.

Abatacept selectively binds to CD80 and CD86, thereby blocking theinteraction with CD28 and interfering with T-cell activation. Itinhibits naive T-cell activation, thus having the potential toselectively inhibit T-cell response to specific antigens instead ofbroad immunosuppression. Effector-memory T-cell responses are lessdependent on CD28 co-stimulation and, presumably, are less inhibited byco-stimulation blockade. (Lo D J, Weaver T A, Stempora L, et al. Am JTransplant 2011; 11: 22-33.)

Studies in both animals and human beings have shown that interruption ofthe co-stimulatory second signal beneficially affects autoimmunityCo-stimulation blockade with abatacept has been shown to have clinicaleffectiveness in psoriasis (Abrams J R, Lebwohl M G, Guzzo C A, et al. JClin Invest 1999; 103: 1243-52) and psoriatic arthritis (Mease P,Genovese M C, Gladstein G, et al. Arthritis Rheum 2011; 63: 939-48) andis approved for treatment of rheumatoid arthritis, Genant H K, Peterfy CG, Westhovens R, et al. Ann Rheum Dis 2008; 67: 1084-89) includingjuvenile rheumatoid arthritis. (Ruperto N, Lovell D J, Quartier P, et alLancet 2008; 372: 383-91.) Additionally, co-stimulation blockade hasbeen effective in control of allograft rejection. (Vincenti F, Larsen C,Durrbach A. N Engl J Med 2005; 353: 770-81.) Moreover, Lenschow andcoworkers (Lenschow D J, Ho S C, Sattar H, et al. J Exp Med 1995; 181:1145-55) showed that costimulatory blockade with both an anti B7-2monoclonal antibody and a CTLA4-immunoglobulin fusion protein preventeddiabetes in the NOD mice model when administered prior to 10 weeks ofage.

It has now been shown that co-stimulation modulation with T-cellco-stimulatory antagonists such as CTLA-4 compositions and in particularabatacept, halts or slows autoimmune β-cell destruction leading topreservation of C-peptide secretion in recently diagnosed patients withType 1 diabetes by blocking the generation of autoaggressive T cells(Orban et al., Lancet 2011; 378 (9789): 412-9.)

Thus, there is provided herein a method of treating, preventing, ordelaying the progression of diabetes mellitus by administering a CTLA4molecule. The methods of the invention can prevent or delay the onset ofdiabetes mellitus, or prevent or delay loss of residual β-cell mass,providing a longer remission period and delaying the onset ofdiabetes-related complications at a later stage of the life.

T1DM may be treated by the methods as described herein. The treatmentcan be for subjects with residual beta-cell function as well as forthose no longer having any beta-cell function. The treatment may also besuggested for subjects provided exogenous beta-cells through transplantor injection or other beta cell replacement modalities (like embryonicor other stem cell therapies or other replacement modalities).

T1DM may be prevented in a subject by first selecting a subject who issusceptible to developing diabetes and administering a CTLA4 molecule asdescribed herein. The subject who is susceptible to developing diabetesmay be selected by the expression of one or more of: GAD65autoantibodies (GAAs), ICA512 autoantibodies (ICA512AAs), oranti-insulin autoantibodies (IAAs). Each of these autoantibodies isassociated with a risk of progression to autoimmune Type 1 diabetes.Expression of two or more of: GAD65 autoantibodies (GAAs), ICA512autoantibodies (ICA512AAs), or anti-insulin autoantibodies (IAAs) isassociated with a high risk of progression to autoimmune Type 1diabetes. (Liping Yu et al., Diabetes August 2001 vol. 50 no. 81735-1740; Verge C F et al., Diabetes 45:926-933, 199; Verge C F. et al,Diabetes 47:1857-1866, 1998; and Bingley P J, et al., Diabetes43:1304-1310, 1994).

The onset of T1DM may be delayed by the methods as described herein suchthat insulin is not needed by the subject for a longer length of time.Alternatively or in addition, the present method may extend the“honeymoon phase” in an already diabetic subject. The honeymoon phase iswhere insulin is secreted by the pancreas, causing high blood sugarlevels to subside, and resulting in normal or near normal glucose levelsdue to responses to insulin injections and treatment.

The CTLA4 molecules as described herein may be administered incombination with a pharmaceutically acceptable carrier and administeredas a pharmaceutical composition. The term “pharmaceutically acceptablecarrier” includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutical compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds ofprovided herein, such as by producing any undesirable biological effector otherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutical composition, its use is contemplatedto be within the scope of this invention. Some examples of materialswhich can serve as pharmaceutically acceptable carriers include, but arenot limited to, sugars such as lactose, glucose and sucrose; starchessuch as corn starch and potato starch; cellulose and its derivativessuch as sodium carboxymethyl cellulose, ethyl cellulose and celluloseacetate; powdered tragacanth; malt; gelatine; talc; excipients such ascocoa butter and suppository waxes; oils such as peanut oil, cottonseedoil; safflower oil, sesame oil; olive oil; corn oil and soybean oil;glycols; such as propylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The compounds described herein including pharmaceutically acceptablecarriers can be delivered to a patient using a wide variety of routes ormodes of administration. Suitable routes of administration include, butare not limited to, inhalation, transdermal, oral, rectal, transmucosal,intestinal and parenteral administration, including intramuscular,subcutaneous and intravenous injections.

The compositions as described herein may be administered with anadjuvant. The term “adjuvant” can be a compound that lacks significantactivity administered alone but can potentiate the activity of anothertherapeutic agent. In some embodiments, an adjuvant is selected from thegroup consisting of buffers, anti-microbial preserving agents,surfactants, antioxidants, tonic regulators, antiseptics, thickeners andviscosity improvers. In some embodiments, the adjuvant is IFA or otheroil-based adjuvant is present between 30-70%, preferably between 40-60%,more preferably between 45-55% proportion weight by weight (w/w). Insome embodiments, CTLA4 and IFA or other oil based adjuvant are presentin about a 50/50 weight by weight ratio. In some embodiments, thepharmaceutical composition is free of contaminants, e.g., pyrogens.

For oral administration, the compound can be formulated readily bycombining the CTLA4 molecule with one or more pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds of the invention to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a patient to be treated. Pharmaceutical preparationsfor oral use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired. to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

The compound may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Injection isa preferred method of administration for the compositions of the currentinvention. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents may be added, such as the cross-linked polyvinyl pyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate. Thus,in some embodiments, the composition may be a water-in-oil emulsion. Inother embodiments, the composition may be an oil-in-water emulsion. Suchoil-in-water emulsions may be particularly useful for controlling therelease profile and providing a slow release of the active drug, whichcan potentially be absorbed unaltered from such an emulsion.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions. For injection, the agents of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

In some embodiments, the composition comprising a CTLA4 molecule alsoincludes an oil-based carrier.

The oil-based carrier is a composition that includes at least 10% byweight of a natural or synthetic oil suitable for administration to ahuman in conjunction with a therapeutic agent. In preferred embodiments,the carrier includes at least 20, 30, 50, 70, 80, 90, 95, 98, or 99% oilby weight. In some embodiments, the oil-based carrier can include lessthan 70, 60, 50, 40, 30 or 20% oil by weight. In preferred embodiments,the oil will be in the range of 10 to 95%, preferably 20 to 90% or 30 to70% oil by weight. The oil should be chosen such that it provides forsustained release of a substance dispersed within it when administeredto a subject. Suitable oils include mineral oil (e.g., Drakeol 6 VRlight mineral oil), vegetable oil, squalene, or liquid paraffin. In someembodiments, the oil-based carrier can contain more than one type ofoil. In some embodiments, the oil-based carrier can include an immunestimulator, e.g., an immunostimulating glucan, but it is much preferredthat the oil-based carrier does not include an immune stimulator, e.g.,an immunostimulating glucan, a bacterial component, e.g., amycobacterial component. In a preferred embodiment, the oil-basedcarrier does not include an alum component.

While not wanting to be bound by theory, an oil based carrier isbelieved to work by triggering the immunocompetent cells, which arerelated to the inflammatory as well as protective ability. An oil-basedcarrier can also act as an antigen vehicle and a slow release orlong-term antigen presentation device. When injected into a subject, anoil-based carrier and antigen composition can form a depot of antigen atthe injection site, thereby protecting the antigen from degradation.From this depot the antigen can be released slowly into the system andprovides a prolonged antigen presentation as well as expanded totalcontact surface area and the attraction of inflammatory cells.Macrophages can digest most of the incorporated materials and presentthe processed antigens on their surface. From this depot the antigen canbe released slowly into the system and provides a prolonged antigensupply to act as costimulatory modulator.

Oil based carriers optionally include an emulsifier or surfactantcomponent. The emulsifier or surfactant (and the amount of emulsifier orsurfactant) is chosen such that it facilitates the mixture or dispersionof a substance, e.g., an antigen preparation, with the oil. An oil-basedcarrier can include 0.1 to 50%, preferably 1 to 30%, more preferably 5to 20% by weight of a surfactant or emulsifier. Examples of emulsifiersor surfactants include Arlacel A, mannide oleate (e.g., Montamide80-mannide monooleate), anhydrous mannitol/oleic acid ester,polyoxyethylene or polyoxypropylene.

An oil-based carrier or adjuvant typically consists of two components:(1) an oil, and (2) an emulsifier or surfactant, mixed with water.Suitable oils and emulsifiers are known in the art. For example, the oilcan be mineral oil, vegetable oil, squalene or liquid paraffin. Theemulsifier or surfactant can be, e.g., Arlacel A, mannide oleate,anhydrous mannitol/oleic acid ester, polyoxyethylene orpolyoxypropylene. Exemplary oil-based adjuvants include conventionalIFA, Montamide ISA adjuvants, or Hunter's TiterMax adjuvant. Inpreferred embodiments, the adjuvant includes 20 to 95%, preferably 30 to90%, more preferably 40 to 70% by weight of an oil phase, and 0.1 to50%, preferably 1 to 30%, more preferably 5 to 20% by weight of asurfactant or emulsifier. Various types of oil-based adjuvants aredescribed, e.g., in U.S. Pat. No. 5,814,321, U.S. Pat. No. 6,299,884,U.S. Pat. No. 6,235,282, and U.S. Pat. No. 5,976,538.

IFA is typically a mixture of a non-metabolizable oil (e g, mineraloil), water, and a surfactant (e.g., Arlacel A). Unlike CompleteFreund's Adjuvant (CFA), IFA does not contain a bacterial component,e.g., mycobacteria. The first large-scale vaccinations using IFA inhumans took place on US military personnel (Davenport (1968) Ann Allergy26:288-292; Beebe et al., (1972) Am J Epidemiol 95:337-346; Salk & Salk(1977) Science 195:834-847). The findings were essentially negative withrespect to malignancy, allergic diseases and collagenosis, but there wasevidence that some men had a cyst like reaction at the site ofinoculation. Follow up studies showed that these adverse events were dueto improper administration of the compound, i.e. it was given s.c.instead of i.m. From these experiments, IFA was regarded by some asunsuitable for human purposes, although it has remained widely used inanimal research. In recent years, newer forms of IFA have been shownsafe for human use in HIV immunotherapy or therapeutic vaccinations(Turner et al. (1994) AIDS 8:1429-1435; Trauger et al. (1995) J AcquirImmune Defic Syndr Hum Retrovirol 10 Supp2:S74-82; Trauger et al. (1994)J Infect Dis 169:1256-1264).

Montanide ISA Adjuvants (Seppic, Paris, France) are a group ofoil/surfactant based adjuvants in which different surfactants arecombined with either a non-metabolizable mineral oil, a metabolizableoil, or a mixture of the two. They are prepared for use as an emulsionwith aqueous Ag solution. The surfactant for Montanide ISA 50(ISA=Incomplete Seppic Adjuvant) is mannide oleate, a major component ofthe surfactant in Freund's adjuvants. The surfactants of the Montanidegroup undergo strict quality control to guard against contamination byany substances that could cause excessive inflammation, as has beenfound for some lots of Arlacel A used in Freund's adjuvant. The variousMontanide ISA group of adjuvants are used as water-in-oil emulsions,oil-in-water emulsions, or water-in-oil-in-water emulsions. Thedifferent adjuvants accommodate different aqueous phase/oil phaseratios, because of the variety of surfactant and oil combinations.

Hunter's TiterMax (CytRx Corp., Norcross, Ga.) is anoil/surfactant-based adjuvant prepared as a water-in-oil emulsion in amanner similar to that used for conventional Freund's adjuvants.However, it uses a metabolizable oil (squalene) and a nonionicsurfactant that has good protein antigen-binding capacity as well asadjuvant activity. The adjuvant activity may relate, in part, to thesurfactant's ability to activate complement and bind complementcomponents, as this helps target the Ag to follicular dendritic cells inthe spleen and lymph nodes. The surfactant used in the commerciallyavailable adjuvant is one of a number of synthetic nonionic blockcopolymers of polyoxyethylene and polyoxypropylene developed by Hunter(Hunter et al., 1991 Vaccine 9:250-256). The utilization ofcopolymer-coated microparticles to stabilize the emulsion permitsformation of stable emulsions with less than 20% oil, an importantfactor in minimizing total adjuvant injected.

An adjuvant can be used with antigens to elicit cell-mediated immunityand the production of antibodies of protective isotypes (IgG2a in miceand IgG1 in primates). Different types of adjuvants share similar sideeffects, such as a reaction at the injection site and pyrogenicity.Alum, a commonly used adjuvant for human vaccine also produces anappreciable granulomatous response at the injection site (Allison &Byars (1991) Mol Immunol 28:279-284). The mode of action of anincomplete Freund's adjuvant can involve non-specific as well asspecific immune responses. IFA seems to work by triggering theimmunocompetent cells, which are related to the inflammatory as well asprotective ability. IFA also acts as an antigen vehicle and a slowrelease or long-term antigen presentation device. Injecting a patientwith an IFA and antigen compound, it forms a depot of antigen at theinjection site, thereby protecting the antigen from degradation. Fromthis depot the antigen is released slowly into the system and provides aprolonged antigen presentation as well as expanded total contact surfacearea and the attraction of inflammatory cells. Macrophages digest mostof the incorporated materials and present the processed antigens ontheir surface. From this depot the antigen can be released slowly intothe system and provides a prolonged antigen supply to act ascostimulatory modulator.

The specific enhancing effect of the IFA on the antigen immunogenicityhas been found to lead to increased humoral immunity (preferentiallyprotective antibody production; IgG1 in humans and IgG2a in mice) and toelicit specific cell mediated immunity (preferentially Th2 type).Specifically, as an example human recombinant insulin B-chain in IFAresults in Th2 cytokine pattern in NOD mice islets (Ramiya et al. (1996)J Autoimmun 9:349-356). IFA is unique among adjuvants tried for diabetesprevention in animal models. Ramiya and coworkers (supra) concluded thatboth alum and DPT as adjuvants have ‘non-specific’ protective effectsunrelated to the antigen used, while IFA is the only one with antigenspecific protective effect for diabetes prevention in animals.

IFA, preferably an IFA approved for human use, e.g., Montanide (e.g.,Montanide ISA51, Seppic Inc., France) or an equivalent composition, is apreferred adjuvant for use in the methods and vaccines described herein.Montanide ISA51 has shown no systemic or significant local side effectsin our animal and in our human studies.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired. to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Injection isa preferred method of administration for the compositions of the currentinvention. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents may be added, such as the cross-linked polyvinyl pyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances, which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents, which increase the solubility of thecompounds to allow for the preparation of highly, concentratedsolutions. For injection, the agents of the invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hanks's solution, Ringer's solution, or physiological salinebuffer. Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The amount of the combination of a CTLA4 molecule provided to thesubject will depend on both the size and weight of the subject as wellas the progression of the disease. For the compounds described herein,the therapeutically effective amount can be initially determined from invitro assays. Since the compounds of the present invention may have alow absorption and low bioavailability, the therapeutically effectiveamount may be determined from, for example, blood level of the compoundsor metabolites thereof or fecal concentration of the compounds ormetabolites thereof. As is well known in the art, therapeuticallyeffective amounts for use in humans can also be determined from animalmodels. A therapeutically effective dose can also be determined fromhuman data for compounds which are known to exhibit similarpharmacological activities. The applied dose can be adjusted based onthe relative potency of the administered compound as compared with theknown compound.

Patient doses for parenteral administration of the compounds describedherein, typically range from about 1 mg/day to about 10,000 mg/day, moretypically from about 10 mg/day to about 1,000 mg/day, and most typicallyfrom about 50 mg/day to about 500 mg/day. Stated in terms of patientbody weight, typical dosages range from about 0.01 to about 150mg/kg/day, more typically from about 0.1 to about 15 mg/kg/day, and mosttypically from about 1 to about 10 mg/kg/day, for example 5 mg/kg/day or3 mg/kg/day.

The CTLA4 molecule may be administered in a single daily dose or it maybe administered multiple times per day. Alternatively, it may beadministered less than once a day. The dosing may be over a period oftime, such as once a month, or every 28 days. In some embodiments,additional doses (e.g., bolus dosing) may be given at the beginning oftreatment. In some embodiments, a dose containing approximately 5, 10,20, 30, 50, 100 mg/kg of the CTLA4 molecule.

The definitions of terms used herein are meant to incorporate thepresent state-of-the-art definitions recognized for each term in thechemical and pharmaceutical fields. Where appropriate, exemplificationis provided. The definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

As used herein, the terms “administering” or “administration” areintended to encompass all means for directly and indirectly delivering acompound to its intended site of action.

The phrase “delaying the progression” as used herein in the context ofdelaying the progression of diabetes mellitus means that the loss offunctional residual β-cell mass, after the clinical onset of Type 1diabetes is delayed. The delayed progression of T1DM can be measured,for example, by measuring C-peptide production.

The phrase “pharmaceutically acceptable” refers to additives orcompositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to an animal, such as a mammal(e.g., a human). The term “pharmaceutically acceptable carrier” includesany and all solvents, diluents, or other liquid vehicle, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, solid binders, lubricants and thelike, as suited to the particular dosage form desired. Remington's, TheScience and Practice of Pharmacy, (Gennaro, A. R., ed., 19^(th) edition,1995, Mack Pub. Co.), discloses various carriers used in formulatingpharmaceutical compositions and known techniques for the preparationthereof. Except insofar as any conventional carrier medium isincompatible with the compounds of provided herein, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatine; talc. Excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil, sesame oil; olive oil; corn oil and soybean oil; glycols; such aspropylene glycol; esters such as ethyl oleate and ethyl laurate; agar;buffering agents such as magnesium hydroxide and aluminum hydroxide;alginic acid; pyrogen-free water; isotonic saline; Ringer's solution;ethyl alcohol, and phosphate buffer solutions, as well as othernon-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

The term “pharmaceutical composition” refers to a composition describedherein, or pharmaceutically acceptable salts thereof, with other agentssuch as carriers and/or excipients. Preferably, a pharmaceuticalcomposition will have the active agent present at least 95% purity, or98% purity, or 99% purity, or more.

As used herein, the term “subject” is a human or other animal, having adiabetes, pre-diabetes, or a predisposition to diabetes. Thus, in someembodiments the subject will be in need of the therapeutic treatment asprovided herein. Preferred patients are mammals. Examples of patientsinclude but are not limited to, humans, horses, monkeys, dogs, cats,mice, rates, cows, pigs, goats and sheep. In some embodiments,“subjects” are generally human patients having diabetes. In someembodiments, “subjects” are human patients who have been diagnosed withT1DM within the last 200, 100, or 50 days. In some embodiments,“subjects” are human patients who have been recently diagnosed withdiabetes mellitus but still have residual beta-cell function. In somesuch embodiments the residual beta-cell function is detectable or atleast 10%, 20%, 30%, 40%, 50%, 60%, or more of the beta cells in a fullyfunctioning pancreas.

The term “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired biological or medicinal response in a cell culture, tissuesystem, animal, or human (e.g., the desired therapeutic result). Atherapeutically effective amount of the composition may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the CTLA4 molecule to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the pharmacologicalagent are outweighed by the therapeutically beneficial effects. In someembodiments, the response includes alleviation and/or delay of onset ofone or more symptoms of the disease, condition, or disorder beingtreated.

The term “treatment” or “treating” as used herein is defined as theapplication or administration of the therapeutic agents to a subject, orapplication or administration of the therapeutic agents to an isolatedtissue or cell line from a subject who has diabetes, a symptom ofdisease or a predisposition toward a disease. Treatment is intended toencompass preventing the onset, slowing the progression, reversing orotherwise ameliorating, improve, or affect the disease, the symptoms orof disease or the predisposition toward disease. For example, treatmentof a subject, e.g., a human subject, with a composition describedherein, can slow, improve, or stop the ongoing autoimmunity, e.g., areaction against pancreatic β-cells, in a subject before, during, orafter the clinical onset of Type 1 diabetes.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined—e.g., the limitations of the measurement system, or thedegree of precision required for a particular purpose. For example,“about” can mean within 1 or within 2 standard deviations, as per thepractice in the art. Alternatively, “about” can mean a range of up to20%, preferably up to 10%, and more preferably up to 5% of a givenvalue. Where particular values are described in the application andclaims, unless otherwise stated, the term “about” meaning within anacceptable error range for the particular value should be assumed.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the,” include plural referents unless the context clearly indicatesotherwise. Thus, for example, reference to “a molecule” includes one ormore of such molecules, “a resin” includes one or more of such differentresins and reference to “the method” includes reference to equivalentsteps and methods known to those of ordinary skill in the art that couldbe modified or substituted for the methods described herein.

While the above description provides examples and specific details ofvarious embodiments, it will be appreciated that some features and/orfunctions of the described embodiments admit to modification withoutdeparting from the scope of the described embodiments. The abovedescription is intended to be illustrative of the invention, the scopeof which is limited only by the language of the claims appended hereto.

EXAMPLES

Aspects of the applicant's teachings may be further understood in lightof the following examples, which should not be construed as limiting thescope of the applicant's teachings in any way.

Example 1—Study Design and Patients

Patients (aged 6-45 years) diagnosed with Type 1 diabetes within thepast 100 days were parallel-screened for this study. Patients wereeligible to participate in the study if they had at least onediabetes-related autoantibody (microassayed insulin antibodies [ifduration of insulin therapy was less than 7 days]; glutamic aciddecarboxylase-65 [GAD-65] antibodies; islet-cell antigen-512 [ICA-512]antibodies; or islet-cell autoantibodies) and had stimulated C-peptideconcentrations of 0.2 nmol/L or higher measured during a mixed-mealtolerance test (MMTT) done at least 21 days after diagnosis of diabetesand within 37 days of randomization.

People whose blood samples screened positive for serum antibodies tohepatitis B surface antigen, hepatitis C, or HIV were excluded fromparticipation. Samples were also tested for Epstein-Barr virus (EBV).Individuals who had evidence of active EBV infection at the time ofscreening were ineligible. Participants who showed evidence of activeEBV infection after randomization did not receive additional study druguntil resolution.

Patients were randomly assigned in a 2:1 ratio, stratified byparticipating site, to receive experimental treatment with abatacept orplacebo using a double blind protocol. Table 1 provides the baselinedemographic and laboratory characteristics of participants

TABLE 1 Abatacept (n = 77) Placebo (n = 35) Age Mean (years) 13.9 (6.9)13.7 (5.3) Median (years) 12 (6-36) 14 (7-34) Men 41 (53%) 25 (71%)Race* White 71 (93%) 32 (91%) Ethnic origin Non-Hispanic 67 (87%) 31(89%) Number of diabetes-related autoantibodies† 1 9 (12 4 (11%) 2 26(34%) 9 (26%) 3 26 (34%) 15 (43%) 4 16 (21%) 7 (20%) Number of days fromdiagnosis 87.9 (14.1) 83.2 (17.8) to first infusion‡ Weight (kg) 52.6(21.9) 53.0 (19.7) Body-mass index (kg/m²) 21.0 (4.5) 20.5 (3.9) MeanAUC for C-peptide (nmol/L) 0.743 (0.42) 0.745 (0.31) HbA1c at baseline*(%) 6.31% (0.80) 6.74% (0.94) Total daily insulin dose at 0.385 (0.24)0.339 (0.22) baseline* (U/kg) Ketoacidosis at diagnosis 25 (32%) 8 (23%)Diabetes-associated HLA alleles present* DR3 and DR4 25 (34%) 16 (49%)DR3 only 11 (15%) 5 (15%) DR4 only 30 (41%) 10 (30%) Neither 8 (11%) 2(6%) Data are n (%), mean (SD), or median (range). AUC = area under thecurve. HbA1c = glycated haemoglobin Alc. *Excludes participants withdata missing for indicated variable (number missing: race, 1; HbAlc, 2;insulin use, 1; HLA allele status, 4). †Islet-cell autoantibodies byimmunofluorescence not tested on 16 patients (considered negative forcount). ‡Range 51-108 for abatacept group and 38-107 for placebo.

Example 2—Procedures

Abatacept (Orencia, Bristol-Myers Squibb, Princeton, N.J., USA) wasgiven on days 1, 14, and 28, and then every 28 days with the last doseon day 700 (total 27 doses) as a 30-min intravenous infusion at a doseof 10 mg/kg (maximum 1000 mg per dose) in a 100 mL 0.9% sodium chlorideinfusion. Normal saline infusion was used as placebo. Patients did notreceive any premedication.

All patients received intensive diabetes management. The goal was toachieve intensive glycaemic control as recommended by the AmericanDiabetes Association. (American Diabetes Association. Diabetes Care2011; 33 (suppl 1): S11-61.) Patients used either multiple daily insulininjections or an insulin pump. Blood glucose monitoring was done bymeans of frequent daily blood glucose monitoring. Use of non-insulinpharmaceuticals that affect glycaemic control was not allowed.

Blood samples were analyzed centrally. C-peptide concentrations weremeasured from frozen plasma with a two-site immunoenzymometric assay(Tosoh Bioscience, South San Francisco, Calif., USA). Glycatedhaemoglobin A1c (HbA1c) was measured with ion-exchange high-performanceliquid chromatography (Variant II, Bio-Rad Diagnostics, Hercules,Calif., USA). Reliability coefficients for each assay were greater than0.99 from split duplicate samples. Biochemical autoantibodies(microassayed insulin antibodies, GAD-65 antibodies, ICA-512 antibodies)were measured with radioimmunobinding assays and islet-cellautoantibodies (ICA) with indirect immunofluorescence. A routinechemistry panel was done (Roche Diagnostics [Indianapolis, Ind., USA]Hitachi 917 Analyzer and reagents). HLA class II alleles were measuredwith PCR amplification and sequence-specific hybridization. β-cellfunction was assessed by stimulated C-peptide secretion. Theprespecified primary outcome of this trial was a comparison of the areaunder the curve (AUC) of stimulated C-peptide response over the first 2h of a 4-h MMTT2, done at the 24-month visit. The 4-h MMTTs were done atbaseline and at 24 months; 2-h MMTTs were obtained at 3, 6, 12, and 18months. Patients who had completed their 2-year visit MMTT were includedin the primary outcome assessment. After completion of the 2-yeartreatment phase, participants entered a 2-year follow-up phase (studyremained double blind) to continue to assess safety and efficacy,including an MMTT every 6 months. Prespecified secondary outcomesincluded: slope of C-peptide over time, difference between groups inincidence of loss of peak C-peptide to less than 0.2 nmol/L, differencesin HbA1c and insulin dose over time, and safety. Prespecified subgroupfactors included age, sex, race, baseline C-peptide, baseline insulinuse, baseline HbA1c, and HLA type.

Example 3—Statistical Analyses

Spotfire S+8.1, a statistical analysis software, was used for allanalyses. A sample size of 108 participants was planned to provide 85%power to detect a 50% increase in geometric mean C-peptide relative tothe placebo group using a test at the 0.05 level (one-sided), with 10%loss to follow-up and a 2:1 allocation to treatment versus control(based on an estimated mean of 0.248 and SD of 0.179, on the transformedscale). All analyses were based on the prespecified intention to treatcohort with known measurements. Missing values were assumed to bemissing at random. The p values associated with the intention-to-treattreatment comparisons of the primary and secondary endpoints aretwo-sided, although the design of the trial was based on a one-sidedhypothesis test. Interim analysis for endpoint treatment effect was doneand reported to the data and safety monitoring board once in accordanceto the method of Lan and DeMets with O'Brien-Fleming boundaries. (Lan KK G, DeMets D L. Biometrika 1983; 70: 659-63.) The prespecified analysismethod for C-peptide mean AUC, HbA1c, and total daily insulin dose wasan analysis of covariance model adjusting for age, sex, and baselinevalue of the dependent variable, and treatment assignment. The predictedmeans and associated 95% confidence intervals (CIs) for each treatmentgroup were established at the means of the other covariates. Thesignificance levels associated with the treatment effect were from theWald test (from the fitted model). A normalizing transformation oflog(XC-Pep+1) was prespecified for C-peptide AUC mean, and normal plotsof the residuals suggested that it was adequate. The C-peptide mean AUCequals the AUC divided by the 2-h interval (i.e., AUC/120). The AUC wascomputed using the trapezoidal rule from the timed measurements ofC-peptide during the MMTT. The time to first stimulated peak C-peptideof less than 0.2 nmol/L (a level above which was associated withdecreased risk of complications) was analyzed with standard survivalmethods (Cox model and Kaplan-Meier method). Adverse event grades wereanalyzed with the Wilcoxon rank sum test. (Agresti A. Categorical dataanalysis. New York, N.Y., USA: John Wiley and Sons, 1990.) Mean rate ofchange of C-peptide mean AUC from 6 to 24 months was estimated with amixed-effects model with both random intercept and slope adjusting forage, sex, baseline C-peptide mean AUC, and treatment assignment. Theinitial fit included a fixed interaction effect of treatment and time,but was removed because of the absence of any statistical evidence of itbeing other than zero. To assess the treatment effect over the entiretime period, we fitted a similar mixed model to the data with thedifferences that we defined time without structure and grouped by6-month intervals.

Example 4—Results

Of the 112 patients enrolled in the study, 77 were randomly assigned toreceive experimental treatment with abatacept and 35 were assigned toreceive placebo. Table 1 summarizes the baseline characteristics of thetwo groups. The only noteworthy imbalances were the greater proportionof men in the placebo group than in the abatacept group and higher meanHbA1c in the placebo group. The number of infusions actuallyadministered by treatment group were compared using a Wilcoxon rank sumtest; no significant difference was detected (p=0.61). Overall, 2514(83%) of 3024 potential infusions were given, and many that were notgiven were per protocol (e.g., patient developed EBV infection or becamepregnant). 689 (93%) of 738 expected MMTTs were done. In the primaryanalysis at 2 years, participants assigned to abatacept had a geometricmean stimulated C-peptide 2-h AUC of 0.375 nmol/L (95% CI 0.290-0.465)versus 0.266 nmol/L (0.171-0.368) for those assigned to placebo. Theadjusted population C-peptide mean 2-h AUC at 2 years was 0.378 nmol/Lfor the abatacept group and 0.238 nmol/L for the placebo group; thus,C-peptide AUC at 2 years was 59% (95% CI 6.1-112) higher with abatacept(p=0.0029). The result remained unchanged and significant (p=0.0028)when baseline HbA1c was added as a covariate. To address the differencein C-peptide concentrations from baseline to the 2-year assessments(primary endpoint), C-peptide results for 3, 6, 12, and 18 months wereseparately modeled.

FIG. 1 shows the adjusted population C-peptide mean 2-h AUC over 2years. Patients who received abatacept had a significantly higher meanAUC at 6, 12, and 18 months than did those assigned to placebo, and overall time points in aggregate (p=0.0022). To calculate the effect oftreatment on delaying the reduction of C-peptide, we calculated thepredicted population mean of C-peptide AUC mean by treatment group overtime (FIG. 2). The lines are based on the fitting of a mixed linearmodel using all available data from MMTTs at 6, 12, 18, and 24 months.When testing for the improvement in the fit for the interaction term ofslope and treatment (i.e., testing the evidence that the two treatmentgroups had differing C-peptide decay rates), this result was notsignificant (p=0.85). Consequently, a simpler model assuming identicalslopes was used and FIG. 2 shows these results. Thus, estimated lag timein the means of the abatacept group to drop to the same level as thoseof the placebo group was 9.6 months (95% CI 3.47-15.6). By the 24-monthassessment, (32%) patients in the abatacept group had an AUC peakstimulated C-peptide less than 0.2 nmol/L, compared with 15 (43%)patients on placebo (FIG. 3). The adjusted relative (abatacept toplacebo groups) risk of peak C-peptide falling below 0.2 nmol/L was0.433 (95% CI 0.218-0.861). During the 24 months of follow-up, theabatacept group had a lower adjusted mean HbA1c (FIG. 4A) than did theplacebo group (for all time points in the aggregate, p=0.002), althoughHbA1c was also lower at baseline. Nonetheless, even after adjustment forthe difference at baseline, the treatment group difference over 24months persists (p=0.0071). At study end, 34 (47%) patients on abatacepthad HbA1c lower than 7% compared with eight (26%) on placebo. This isparticularly noteworthy as 86% of all patients were under 18 years ofage; in this group this HbA1c is better that the ADA age-specific targetHbA1c. Participants in the abatacept group had lower insulin doses atsome time points (6 and 12 months) during the study, but at 24 months,insulin doses in the two groups were similar (FIG. 4; p=NS at 24 months,but because of differences at the earlier time points, p=0.040 for alltime points in the aggregate).

FIG. 5 shows the results of a homogeneity test of treatment effect onage, sex, race, baseline C-peptide, baseline insulin use, baselineHbA1c, and HLA type. The apparent adverse effect of abatacept innon-white participants might be hypothesis-generating, however thegroups size was small.

Table 2 and Table 3 summarize safety and adverse events. Abatacept waswell tolerated. Infusion-related adverse events occurred with lowfrequency (47 of 2514 infusions [2%] involving 27 patients) and were notclinically significant. Of these, 36 reactions occurred in 17 (22%) of77 patients on abatacept and 11 reactions in six (17%) of 35 patients onplacebo (p=0.62 for proportion of participants by Fisher's exact test).Overall adverse event rate (including laboratory abnormalities) was lowwith no difference between the two groups. Specifically, there was noincrease in infection (including EBV) or in neutropenia (which occurredin seven [9%] of patients on abatacept, five [14%] on placebo). Therewere seven episodes of hypoglycemia reported as an adverse event, two ofwhich were severe hypoglycemia (one in each group).

TABLE 2 Number of patients by worst grade of adverse effects Abatacept(n = 77) Placebo (n = 35) None 14 (18%)  8 (23%) Grade 1 1 (1%) 1 (3%)Grade 2 44 (57%) 17 (49%) Grade 3 12 (16%)  7 (20%) Grade 4 5 (6%) 2(6%) Grade 5 1 (1%)* 0 Data are n (%). Worst grade by treatment groupwas not statistically different with a Wilcoxon Rank Sum Test.*Accidental death, unrelated to study.

TABLE 3 Number of events and patients by type of adverse event PlaceboPlacebo Abatacept (n = 35) Abatacept (n = 35) (n = 77) Number of (n =77) Number of Number of patients with Number of patients with eventsevents events events Allergy/ 3 2 (3%) 0 0 immunology Auditory/ear 3 3(4%) 0 0 Blood/bone marrow 16 11 (14%) 18  6 (17%) Cardiac arrhythmia 11 (1%) 1 1 (3%) Cardiac, general 2 2 (3%) 0 0 Constitutional 19 15 (19%)2 2 (6%) symptoms Death* 1 1 (1%) 0 0 Dermatology/skin 15 13 (17%) 5  4(11%) Endocrine 4 4 (5%) 2 2 (6%) Gastrointestinal 30 18 (23%) 11  7(20%) Infection 63 32 (42%) 31 15 (43%) Hypoglycaemia 5 3 (4%) 2 1 (3%)Metabolic/ 8 6 (8%) 4 2 (6%) laboratory† Musculoskeletal/ 13 11 (14%) 7 6 (17%) soft tissue Neurology 13  8 (10%) 3 2 (6%) Ocular/visual 3 3(4%) 1 1 (3%) Pain 7 6 (8%) 5  4 (11%) Pulmonary/upper 20 10 (13%) 7  4(11%) respiratory Renal/genitourinary 0 0 1 1 (3%) Secondary 1 1 (1%) 00 malignancy Sexual/reproductive 1 1 (1%) 0 0 function Surgery/ 2 2 (3%)0 0 intraoperative injury Syndromes 9  9 (12%) 5  5 (14%) Total 239 105Data are n or n (%). Adverse effect category by treatment group wastested with a one-sided (alternative of higher frequency in abataceptgroup) Fisher's Exact Test; only constitutional symptoms weresignificant (p = 0·049). *Accidental death, unrelated to study. †Otherthan hypoglycaemia.

Example 5—Discussion

Results show that over 2 years co-stimulation modulation with abataceptslows the reduction in β-cell function in recent-onset Type 1 diabetesby 9.6 months. The early beneficial effect suggests that T-cellactivation still occurs around the time of clinical diagnosis of Type 1diabetes, even though the disease course has presumably been in progressfor several years. However, despite continued administration ofabatacept over 24 months, the fall in β-cell function in the abataceptgroup parallels that in the placebo group on the basis of themixed-model results that included the time interval from 6 to 24 months.This subsequent reduction in β-cell function causes us to speculate thatcontinuing T-cell activation subsides as the clinical course of thedisease progresses. Nevertheless, the difference from the placebo groupis maintained during drug administration. Further observation willestablish whether the beneficial effect continues after cessation ofmonthly abatacept infusions. Follow up of these patient shows that thedrug beneficial effect lasts beyond the drug administration for at leastone year.

Abatacept was well tolerated, with no difference between the two groupsin adverse events. However, a potential limitation to clinicalapplicability is that live vaccines cannot be used within 3 months ofabatacept treatment. This factor might be important in view of the youngage of the target population. The main effect seems to occur early afterinitiation of treatment with subsequent resumption of the fall in β-cellfunction. This pattern is reminiscent of the effects of anti-CD3,anti-CD20, and a GAD-65 vaccine, all of which showed some efficacyfollowed later by a reduction in β-cell function parallel to that in thecontrol group. However this approach stands out as this has little or noappreciable side effects unlike the other interventions enlisted. Thisfinding is consistent with our notion that there is an early window ofopportunity after diagnosis in which T-cell activation is prominent. The59% higher mean AUC C-peptide with abatacept than with placebo at 24months in our study is similar to that seen with those otherinterventions, although direct comparison of studies is difficultbecause of differences in important baseline characteristics, includingage, disease duration at time of randomization, and baseline HbA1c.Moreover, our study differs from those studies in that abatacept wasadministered continuously throughout the study, whereas in the case ofanti-CD3, anti-CD20, and GAD-65 vaccine, administration of drug wascompleted within 2-4 weeks after randomization. Crucially, our study wasnot designed to establish whether a short treatment protocol would besufficient to maintain improved C-peptide secretion over 2 years orwhether a continuation of treatment is needed beyond 2 years. With allpatients having completed their course of abatacept, the ongoingfollow-up phase of the study will investigate whether the improvedC-peptide secretion is sustained after discontinuation of the drug andfor how long. Long-term follow-up of patients in one anti-CD3 trialshowed diminishing difference in C-peptide secretion between the treatedand the placebo group after 3 years. This is not the case for abataceptas the data one year off treatment shows that the beneficial effect ismaintained, the difference in C-peptide preservation between theabatacept treated and the placebo group has not diminished (62% moreC-peptide in the abatacept group at 3 years than the placebo group). Inaddition, the abatacept treated group HbA1c remained significantlybetter even after one year off treatment.

In the abatacept group, mean HbA1c was lower than that in the placebogroup throughout the trial, although it was also lower at baseline. Themaintenance of HbA1c lower than 7% for 18 months in theabatacept-treated group is noteworthy because 96 (86%) studyparticipants were 18 years or younger. The clinical importance of HbA1cat this improved level has been well documented. (The Diabetes Controland Complications Trial Research Group. N Engl J Med 1993; 329: 977-86.)Insulin use was similar in the two groups and thus did not contribute tothe difference in HbA1c. In our trial, abatacept-treated patients withrecent-onset Type 1 diabetes had more endogenous insulin production,measured by C-peptide, during the 2 years of study drug administration.The duration of these effects after discontinuation of abatacept isbeing tested in ongoing follow-up of these patients. Theone-year-off-therapy data shows that the beneficial effects of abataceptpersist at least for one year beyond drug administration. The patientsare being followed further. Abatacept administered over 2 years showedan excellent safety profile in patients with Type 1 diabetes. Its maineffect seems to occur early after the initiation of treatment, howeverfurther studies are needed to test how far in the autoimmune processthis drug can be effective in slowing down the autoimmunity. Theseapproaches might be more easily tested with a subcutaneous version ofabatacept.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described inany way.

While the applicant's teachings are described in conjunction withvarious embodiments, it is not intended that the applicant's teachingsbe limited to such embodiments. On the contrary, the applicant'steachings encompass various alternatives, modifications, andequivalents, as will be appreciated by those of skill in the art.

The invention claimed is:
 1. A method of treating type 1 diabetesmellitus in a subject comprising administering an effective amount of afusion protein composition which comprises a fusion protein and anoil-based carrier, wherein the fusion protein comprises theextracellular domain of CTLA4 and a portion of an immunoglobulinmolecule, and wherein the oil-based carrier is a water-in-oil emulsion.2. The method of claim 1 wherein the composition comprises Abatacept. 3.The method of claim 1 wherein the composition is administered as apharmaceutically acceptable salt.
 4. The method of claim 1, wherein thecomposition is administered by intravenous infusion.
 5. The method ofclaim 4, wherein the composition is administered by intravenous infusionin about 50 to 200 ml of physiological saline.
 6. The method of claim 4,wherein the composition is administered at a dose ranging from about 5mg/kg to about 50 mg/kg.
 7. The method of claim 4, wherein theintravenous infusion of the composition is repeated over time.
 8. Themethod of claim 4, wherein the intravenous infusion of the compositionis repeated at least once following a time interval ranging from aboutone week to about two months.
 9. The method of claim 4, wherein thecomposition is administered at a dose ranging from about 250 to 2000 mg.10. The method of claim 9, wherein the composition is administered at adose of 500 mg.
 11. The method of claim 9, wherein the composition isadministered at a dose of 750 mg.
 12. The method of claim 9, wherein thecomposition is administered at a dose of 1000 mg.
 13. The method ofclaim 1, wherein the subject is white.